Cryptosporidium parvum causes one of the opportunistic infections among AIDS patients for which no treatment is yet available. The development of C. parvum consists of several distinct cell cycles referred to as sporogony, merogony, gametogony and oocyst formation. During sporogony and merogony, parasites undergo multiplication to form 4 to 8 daughter cells in each cell cycle, which differs from the duplicative cell cycle in its hosts, and suggests that a unique mechanism may regulate apicomplexan multiplications. Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA)-binding protein, consisting of 3 subunits and playing multiple roles in the DNA replication, repair, and recombination. We have previously discovered a short-type C. parvum RPA large subunit (CpRPA1A, 54 kDa) that differs from that of humans (approximately 70 kDa). More recently, a second RPA1 homologue (CpRPA1 B) was identified from the parasite that differs from CpRPA1A. Since humans have only one RPA1 protein, the presence of two distinct CpRPA1A & 1B proteins in Cp, together with the fact that both CpRPA1A & 1B lack the N-terminal regulatory domain, lead us to hypothesize that C. parvum not only possesses unique DNA replication proteins, but possibly also differs from its host in the regulation of DNA metabolism. Since the growth and development of all organisms depend on the faithful replication of DNA replication in each cell cycle, the peculiar DNA replication machinery in the parasite may be pursued as a novel drug target against cryptosporidiosis. Our long-term goal is to elucidate the structure and function of Cp replication proteins associated with the unique parasite cell and life cycles for chemotherapeutic exploration. The specific aim for the project is to investigate the expression, posttranslational processing and functional properties of CpRPA1A and CpRPA1B genes using proteomics, metabolic labeling, molecular engineering, immunolabeling, confocal and electron microscopy, and a Toxoplasma gondii transfection system. RPA proteins have been promising chemotherapeutic targets against various cancers. The apparent differences between human and C. parvum RPA proteins and their functions support the notion that the parasite RPA may serve as a potential drug target. The completion of these aims will not only prove or refute our hypothesis, but also establish a new foundation for the drug discovery targeting the parasite unique DNA mechanism machinery.